Our studies have implicated a role for protein 4.1R (4.1R) in cell division. Our goal is to understand how 4.1 R is involved in the process of building the mitotic spindle necessary for chromosome segregation. We propose to elucidate the mechanism by which 4.1 R is involved in the regulation of microtubule assembly because microtubules are critical for forming mitotic spindles and because our recent work suggests a role for 4.1R isoforms in both microtubule dynamics and mitotic spindle assembly. We have identified two microtubule associated 4.1R isoforms (4.1R80deltaE5 and 4.1 R135) that interact with microtubule polymers and tubulin dimers. Each has distinct functions. 4.1R80deltaE5 destabilizes interphase microtubules. The 4.1R135 isoform, on the other hand, promotes the organization of microtubules into mitotic asters in an in vitro assembly assay. Moreover, 4.1R135 is phosphorylated by cdc2 kinase and localizes to the mitotic spindle/spindle poles. Overexpression or knockdown of 4.1R impairs cell proliferation. Our proposed studies exploit the tools developed in the previous grant period to address 4.1R's role in the regulation of microtubule and mitotic spindle assembly. We shall use both a model HeLa cell system and a well characterized G1E-ER proliferating erythroblast line in which 4.1R function can be studied during synchronized terminal differentiation. We shall dissect the molecular mechanisms by which 4.1R and its associated proteins, NuMA, dynein, and dynactin regulate mitotic spindle organization, using in vitro and in vivo biochemical assays, fluorescence-based imaging methods. Functional perturbations will be achieved by utilizing siRNA, a Tet-off expression system, and micro-injection of function-blocking proteins or antibodies. We shall also study the role played by 4.1R in microtubule dynamics. To this end, we shall analyze the interaction of 4.1 R domains with alpha/beta tubulin heterodimers and microtubules in order to access its impact on assembly dynamics. In addition, we shall examine the regulation of this interaction during the cell cycle, and the mechanisms by which 4.1R80deltaE5 destabilizes microtubules. Finally, we shall define the relationship between changes in the expression of these 4.1R isoforms and the transition from proliferation to post-mitotic states during erythroid differentiation.